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1.
Microb Cell Fact ; 20(1): 87, 2021 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-33882914

RESUMEN

Trans-4-hydroxy-L-proline is an important amino acid that is widely used in medicinal and industrial applications, particularly as a valuable chiral building block for the organic synthesis of pharmaceuticals. Traditionally, trans-4-hydroxy-L-proline is produced by the acidic hydrolysis of collagen, but this process has serious drawbacks, such as low productivity, a complex process and heavy environmental pollution. Presently, trans-4-hydroxy-L-proline is mainly produced via fermentative production by microorganisms. Some recently published advances in metabolic engineering have been used to effectively construct microbial cell factories that have improved the trans-4-hydroxy-L-proline biosynthetic pathway. To probe the potential of microorganisms for trans-4-hydroxy-L-proline production, new strategies and tools must be proposed. In this review, we provide a comprehensive understanding of trans-4-hydroxy-L-proline, including its biosynthetic pathway, proline hydroxylases and production by metabolic engineering, with a focus on improving its production.


Asunto(s)
Bacterias/metabolismo , Hidroxiprolina/biosíntesis , Ingeniería Metabólica/métodos
2.
Lett Appl Microbiol ; 72(1): 53-59, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32955742

RESUMEN

A new trans-4-hydroxy-l-proline (trans-Hyp) producing Bacillus cereus HBL-AI, was isolated from the air, which was screened just using l-proline as carbon and energy sources. This strain exhibited 73·4% bioconversion rate from initial l-proline (3 g l-1 ) to trans-Hyp. By sequencing the genome of this bacterium, 6244 coding sequences were obtained. Genome annotation analysis and functional expression were used to identify the proline-4-hydroxylase (BP4H) in HBL-AI. This enzyme belonged to a family of 2-oxoglutarate-related dioxygenases, which required 2-oxoglutarate and O2 as co-substrates for the reaction. Homologous modelling indicated that the enzyme had two monomers and contained conserved motifs, which included a distorted 'jelly roll' ß strand core and the residues (HXDXnH and RXS). The engineering Escherichia coli 3 Δ W3110/pTrc99a-proba-bp4h was constructed using BP4H, which transformed glucose to trans-Hyp in one step with high concentration of 46·2 g l-1 . This strategy provides a green and efficient method for synthesis of trans-Hyp and thus has a great potential in industrial application.


Asunto(s)
Bacillus cereus/enzimología , Genoma Bacteriano/genética , Hidroxiprolina/biosíntesis , Prolil Hidroxilasas/metabolismo , Bacillus cereus/genética , Bacillus cereus/aislamiento & purificación , Bacillus cereus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosa/metabolismo , Ácidos Cetoglutáricos/metabolismo , Anotación de Secuencia Molecular , Prolina/metabolismo , Prolil Hidroxilasas/genética
3.
Appl Microbiol Biotechnol ; 104(11): 4771-4779, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32291491

RESUMEN

Hydroxyproline is an industrially important compound with applications in the pharmaceutical, nutrition, and cosmetic industries. trans-4-Hydroxy-L-proline is recognized as the most abundant of the eight possible isomers (hydroxy group at C-3 or C-4, cis- or trans-configuration, and L- or D-form). However, little attention has been paid to the rare isomers, probably due to their limited availability. This mini-review provides an overview of recent advances in microbial and enzymatic processes to develop practical production strategies for various hydroxyprolines. Here, we introduce three screening strategies, namely, activity-, sequence-, and metabolite-based approaches, allowing identification of diverse proline-hydroxylating enzymes with different product specificities. All naturally occurring hydroxyproline isomers can be produced by using suitable hydroxylases in a highly regio- and stereo-selective manner. Furthermore, crystal structures of relevant hydroxylases provide much insight into their functional roles. Since hydroxylases acting on free L-proline belong to the 2-oxoglutarate-dependent dioxygenase superfamily, cellular metabolism of Escherichia coli coupled with a hydroxylase is a valuable source of 2-oxoglutarate, which is indispensable as a co-substrate in L-proline hydroxylation. Further, microbial hydroxyproline 2-epimerase may serve as a highly adaptable tool to convert L-hydroxyproline into D-hydroxyproline. KEY POINTS: • Proline hydroxylases serve as powerful tools for selectivel-proline hydroxylation. • Engineered Escherichia coli are a robust platform for hydroxyproline production. • Hydroxyproline epimerase convertsl-hydroxyproline intod-hydroxyproline.


Asunto(s)
Escherichia coli/enzimología , Hidroxiprolina/biosíntesis , Isomerasas de Aminoácido/metabolismo , Biocatálisis , Inducción Enzimática , Hidroxilación , Isomerismo , Oxigenasas de Función Mixta/metabolismo
4.
Appl Microbiol Biotechnol ; 103(14): 5689-5698, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31106391

RESUMEN

L-Hydroxyproline (Hyp) is a valuable intermediate for the synthesis of pharmaceuticals; consequently, a practical process for its production has been in high demand. To date, industrial processes have been developed by using L-Pro hydroxylases. However, a process for the synthesis of trans-3-Hyp has not yet been established, because of the lack of highly selective enzymes that can convert L-Pro to trans-3-Hyp. The present study was designed to develop a biocatalytic trans-3-Hyp production process. We speculated that ectoine hydroxylase (EctD), which is involved in the hydroxylation of the known compatible solute ectoine, may possess the ability to hydroxylate L-Pro, since the structures of ectoine and 5-hydroxyectoine resemble those of L-Pro and trans-3-Hyp, respectively. Consequently, we discovered that ectoine hydroxylases from Halomonas elongata, as well as some actinobacteria, catalyzed L-Pro hydroxylation to form trans-3-Hyp. Of these, ectoine hydroxylase from Streptomyces cattleya also utilized 3,4-dehydro-L-Pro, 2-methyl-L-Pro, and L-pipecolic acid as substrates. In the whole-cell bioconversion of L-Pro into trans-3-Hyp using Escherichia coli expressing the ectD gene from S. cattleya, only 12.4 mM trans-3-Hyp was produced from 30 mM L-Pro, suggesting a rapid depletion of 2-oxoglutarate, an essential component of enzyme activity as a cosubstrate, in the host. Therefore, the endogenous 2-oxoglutarate dehydrogenase gene was deleted. Using this deletion mutant as the host, trans-3-Hyp production was enhanced up to 26.8 mM from 30 mM L-Pro, with minimal loss of 2-oxoglutarate. This finding is not only beneficial for trans-3-Hyp production, but also for other E. coli bioconversion processes involving 2-oxoglutarate-utilizing enzymes.


Asunto(s)
Halomonas/enzimología , Hidroxiprolina/biosíntesis , Oxigenasas de Función Mixta/metabolismo , Prolina/metabolismo , Streptomyces/enzimología , Aminoácidos Diaminos , Proteínas Bacterianas/metabolismo , Biocatálisis , Escherichia coli/genética , Eliminación de Gen , Hidroxilación , Complejo Cetoglutarato Deshidrogenasa/genética
5.
Environ Toxicol ; 34(6): 719-727, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30810263

RESUMEN

Nickel oxide nanoparticles (Nano NiO) could induce pulmonary fibrosis, however, the mechanisms are still unknown. The aim of the present study was to explore the roles of transforming growth factor-ß1 (TGF-ß1), mitogen-activated protein kinase (MAPK) pathway and MMPs/TIMPs balance in Nano NiO-induced pulmonary fibrosis. For that purpose, we first established Nano NiO-induced human lung adenocarcinoma epithelial cells (A549 cells) model of collagen excessive formation through detecting the levels of hydroxyproline (Hyp) and type I collagen (Col-I). Then the protein levels of TGF-ß1, MAPKs, and MMPs/TIMPs were assessed by Western blot. The results showed that Nano NiO resulted in the increased contents of Hyp, Col-I, and TGF-ß1, the MAPK pathway activation and MMPs/TIMPs imbalance with a dose-dependent manner. In addition, to investigate whether TGF-ß1 mediated MAPK signaling pathway, A549 cells were treated by 100 µg/mL Nano NiO combined with TGF-ß1, p38 MAPK, and ERK1/2 inhibitors (10 µM SB431542, 10 µM SB203580, and 10 µM U0126), respectively. We found that MAPK signal pathway was suppressed by TGF-ß1 inhibitor. Meanwhile, the increased contents of Hyp and Col-I, and MMPs/TIMPs imbalance were alleviated by the p38 MAPK and ERK1/2 inhibitors, respectively. These findings indicated that the MAPK pathway and MMPs/TIMPs imbalance were involved in collagen excessive formation induced by Nano NiO.


Asunto(s)
Colágeno Tipo I/biosíntesis , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Nanopartículas/toxicidad , Níquel/toxicidad , Factor de Crecimiento Transformador beta1/metabolismo , Células A549 , Humanos , Hidroxiprolina/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fibrosis Pulmonar/inducido químicamente
6.
Amino Acids ; 50(1): 29-38, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-28929384

RESUMEN

Glycine, proline, and hydroxyproline (Hyp) contribute to 57% of total amino acids (AAs) in collagen, which accounts for one-third of proteins in animals. As the most abundant protein in the body, collagen is essential to maintain the normal structure and strength of connective tissue, such as bones, skin, cartilage, and blood vessels. Mammals, birds, and fish can synthesize: (1) glycine from threonine, serine, choline, and Hyp; (2) proline from arginine; and (3) Hyp from proline residues in collagen, in a cell- and tissue-specific manner. In addition, livestock (e.g., pigs, cattle, and sheep) produces proline from glutamine and glutamate in the small intestine, but this pathway is absent from birds and possibly most fish species. Results of the recent studies indicate that endogenous synthesis of glycine, proline, and Hyp is inadequate for maximal growth, collagen production, or feed efficiency in pigs, chickens, and fish. Although glycine, proline and Hyp, and gelatin can be used as feed additives in animal diets, these ingredients except for glycine are relatively expensive, which precludes their inclusion in practical rations. Alternatively, hydrolyzed feather meal (HFM), which contains 9% glycine, 5% Hyp, and 12% proline, holds great promise as a low cost but abundant dietary source of glycine, Hyp, and proline for ruminants and nonruminants. Because HFM is deficient in most AAs, future research efforts should be directed at improving the bioavailability of its AAs and the balance of AAs in HFM-supplemented diets. Finally, HFM may be used as a feed additive to prevent or ameliorate connective tissue disorders in domestic and aquatic animals.


Asunto(s)
Fenómenos Fisiológicos Nutricionales de los Animales , Colágeno/biosíntesis , Glicina/metabolismo , Hidroxiprolina/metabolismo , Prolina/metabolismo , Alimentación Animal/análisis , Alimentación Animal/economía , Animales , Colágeno/química , Alimentos Fortificados/análisis , Alimentos Fortificados/economía , Glicina/biosíntesis , Glicina/química , Hidroxiprolina/biosíntesis , Hidroxiprolina/química , Redes y Vías Metabólicas , Prolina/biosíntesis , Prolina/química , Especificidad de la Especie
7.
Lett Appl Microbiol ; 66(5): 400-408, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29432647

RESUMEN

Trans-4-Hydroxy-l-proline (trans-Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Bioconversion of l-proline using recombinant strain with proline-4-hydroxylase (P4H) is a preferred biocatalytic process in the economical production of trans-Hyp. In this study, a recombinant E. coli overexpressing hydroxylase (P4H), γ-glutamyl kinase and glutamate-semialdehyde dehydrogenase (ProBA) genes were constructed by knocking out the key genes in the metabolism. These key genes contained putA encoding proline dehydrogenase (PutA) in the l-proline metabolism and other catalytic enzyme genes, sucAB encoding α-ketoglutarate dehydrogenase (SucAB), aceAK encoding isocitratelyase (AceA) and isocitrate dehydrogenase kinase/phosphatase (AceK) in the TCA cycle. This recombinant strain coupled the synthetic pathway of trans-Hyp with TCA cycle of the host strain. It inhibited the consumption of l-proline completely and promoted the accumulation of 2-oxoglutarate (2-OG) as a co-substrate, which realized the highest conversion of glucose to trans-Hyp. A fed-batch strategy was designed, capable of producing 31·0 g l-1 trans-Hyp from glucose. It provided a theoretical basis for commercial conversion of glucose to trans-Hyp. SIGNIFICANCE AND IMPACT OF THE STUDY: Trans-4-Hydroxy-l-proline (trans-Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Unsatisfactory microbial bioconversion resulted in a low yield of trans-Hyp. In this study, we blocked the unwanted blunting pathways of host strain and make the cell growth couple with the trans-Hyp synthesis from glucose. Finally, a recombinant Escherichia coli with short-cut and efficient trans-Hyp biosynthetic pathway was obtained. It provided a theoretical basis for commercial production of trans-Hyp.


Asunto(s)
Escherichia coli , Glucosa/metabolismo , Hidroxiprolina/biosíntesis , Ingeniería Metabólica/métodos , Prolina/metabolismo , Biocatálisis , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Glutamato-5-Semialdehído Deshidrogenasa/genética , Glutamato-5-Semialdehído Deshidrogenasa/metabolismo , Hidroxiprolina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Fosfotransferasas (aceptor de Grupo Carboxilo)/genética , Fosfotransferasas (aceptor de Grupo Carboxilo)/metabolismo , Prolil Hidroxilasas/genética , Prolil Hidroxilasas/metabolismo
8.
Biotechnol Bioeng ; 114(7): 1511-1520, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28266022

RESUMEN

Amino acid hydroxylases depend directly on the cellular TCA cycle via their cosubstrate α-ketoglutarate (α-KG) and are highly useful for the selective biocatalytic oxyfunctionalization of amino acids. This study evaluates TCA cycle engineering strategies to force and increase α-KG flux through proline-4-hydroxylase (P4H). The genes sucA (α-KG dehydrogenase E1 subunit) and sucC (succinyl-CoA synthetase ß subunit) were alternately deleted together with aceA (isocitrate lyase) in proline degradation-deficient Escherichia coli strains (ΔputA) expressing the p4h gene. Whereas, the ΔsucCΔaceAΔputA strain grew in minimal medium in the absence of P4H, relying on the activity of fumarate reductase, growth of the ΔsucAΔaceAΔputA strictly depended on P4H activity, thus coupling growth to proline hydroxylation. P4H restored growth, even when proline was not externally added. However, the reduced succinyl-CoA pool caused a 27% decrease of the average cell size compared to the wildtype strain. Medium supplementation partially restored the morphology and, in some cases, enhanced proline hydroxylation activity. The specific proline hydroxylation rate doubled when putP, encoding the Na+ /l-proline transporter, was overexpressed in the ΔsucAΔaceAΔputA strain. This is in contrast to wildtype and ΔputA single-knock out strains, in which α-KG availability obviously limited proline hydroxylation. Such α-KG limitation was relieved in the ΔsucAΔaceAΔputA strain. Furthermore, the ΔsucAΔaceAΔputA strain was used to demonstrate an agar plate-based method for the identification and selection of active α-KG dependent hydroxylases. This together with the possibility to waive selection pressure and overcome α-KG limitation in respective hydroxylation processes based on living cells emphasizes the potential of TCA cycle engineering for the productive application of α-KG dependent hydroxylases. Biotechnol. Bioeng. 2017;114: 1511-1520. © 2017 Wiley Periodicals, Inc.


Asunto(s)
Ciclo del Ácido Cítrico/genética , Escherichia coli/fisiología , Mejoramiento Genético/métodos , Hidroxiprolina/biosíntesis , Ácidos Cetoglutáricos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Catálisis , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Hidroxiprolina/genética , Oxigenasas de Función Mixta/genética , Ingeniería de Proteínas/métodos
9.
BMC Biotechnol ; 14: 44, 2014 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-24885047

RESUMEN

BACKGROUND: Trans-4-hydroxy-L-proline (trans-Hyp), one of the hydroxyproline (Hyp) isomers, is a useful chiral building block in the production of many pharmaceuticals. Although there are some natural biosynthetic pathways of trans-Hyp existing in microorganisms, the yield is still too low to be scaled up for industrial applications. Until now the production of trans-Hyp is mainly from the acid hydrolysis of collagen. Due to the increasing environmental concerns on those severe chemical processes and complicated downstream separation, it is essential to explore some environment-friendly processes such as constructing new recombinant strains to develop efficient process for trans-Hyp production. RESULT: In this study, the genes of trans-proline 4-hydroxylase (trans-P4H) from diverse resources were cloned and expressed in Corynebacterium glutamicum and Escherichia coli, respectively. The trans-Hyp production by these recombinant strains was investigated. The results showed that all the genes from different resources had been expressed actively. Both the recombinant C. glutamicum and E. coli strains could produce trans-Hyp in the absence of proline and 2-oxoglutarate. CONCLUSIONS: The whole cell microbial systems for trans-Hyp production have been successfully constructed by introducing trans-P4H into C. glutamicum and E. coli. Although the highest yield was obtained in recombinant E. coli, using recombinant C. glutamicum strains to produce trans-Hyp was a new attempt.


Asunto(s)
Corynebacterium glutamicum/metabolismo , Escherichia coli/metabolismo , Hidroxiprolina/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/enzimología , Escherichia coli/enzimología , Hidroxiprolina/química , Isomerismo , Prolil Hidroxilasas/genética , Prolil Hidroxilasas/metabolismo
10.
J Biol Chem ; 287(44): 37134-44, 2012 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-22930750

RESUMEN

Hypoxia-inducible factors (HIFs) are the master regulators of hypoxia-responsive genes. They play a critical role in the survival, development, and differentiation of chondrocytes in the avascular hypoxic fetal growth plate, which is rich in extracellular matrix (ECM) and in its main component, collagens. Several genes involved in the synthesis, maintenance, and degradation of ECM are regulated by HIFs. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen synthesis because the resulting 4-hydroxyprolines are necessary for the stability of all collagen molecules. The vertebrate C-P4Hs are α(2)ß(2) tetramers with three isoforms of the catalytic α subunit, yielding C-P4Hs of types I-III. C-P4H-I is the main form in most cells, but C-P4H-II is the major form in chondrocytes. We postulated here that post-translational modification of collagens, particularly 4-hydroxylation of proline residues, could be one of the modalities by which HIF regulates the adaptive responses of chondrocytes in fetal growth plates. To address this hypothesis, we used primary epiphyseal growth plate chondrocytes isolated from newborn mice with conditionally inactivated genes for HIF-1α, HIF-2α, or the von Hippel-Lindau protein. The data obtained showed that C-P4H α(I) and α(II) mRNA levels were increased in hypoxic chondrocytes in a manner dependent on HIF-1 but not on HIF-2. Furthermore, the increases in the C-P4H mRNA levels were associated with both increased amounts of the C-P4H tetramers and augmented C-P4H activity in hypoxia. The hypoxia inducibility of the C-P4H isoenzymes is thus likely to ensure sufficient C-P4H activity for collagen synthesis occurring in chondrocytes in a hypoxic environment.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Condrocitos/enzimología , Placa de Crecimiento/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Hipoxia de la Célula/genética , Células Cultivadas , Condrocitos/metabolismo , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Hidroxiprolina/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Cultivo Primario de Células , Procolágeno-Prolina Dioxigenasa/metabolismo , Multimerización de Proteína , Transcripción Genética , Activación Transcripcional , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo
11.
Lipids Health Dis ; 12: 46, 2013 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-23547923

RESUMEN

BACKGROUND: Delayed wound healing is considered one of the most serious diabetes-associated complications. The presence of replicating organisms such as bacteria within a diabetic's wound is considered one of the most important factors that impair cutaneous wound healing and the potential cellular and/or molecular mechanisms that are involved in the healing process. Defensins, which are anti-microbial peptides, have potent bactericidal activity against a wide spectrum of the bacterial and fungal organisms that are commonly responsible for wound infections. We recently demonstrated that camel whey proteins (WPs) expedite the healing of diabetic wounds by enhancing the immune response of wounded tissue cells and by alleviating some of the diabetic complications. METHODS: In the present study, we investigated the effects of WP supplementation on the mRNA and protein expression levels of ß-defensin-1 (BD-1), 2 and 3 and subsequently on the wound healing process in a streptozotocin (STZ)-induced diabetic mouse model. In this study, three groups of mice were used (10 mice per group): group 1, the non-diabetic mice (control); group 2, the diabetic mice; and group 3, the diabetic mice that received a daily supplement of undenatured WP (100 mg/kg of body weight) via oral gavage for 1 month. RESULTS: Compared with the non-diabetic control mice, the diabetic mice exhibited delayed wound closure that was characterized by a reduction in hydroxyproline content (indicator of collagen deposition), a marked elevation in free radical levels and a prolonged elevation in the levels of inflammatory cytokines, including interleukin-6 (IL-6), transforming growth factor-beta (TGF-ß) and tumor necrosis factor-alpha (TNF-α). Interestingly, compared with the diabetic mice that did not receive WP supplementation, the diabetic mice with WP had an accelerated closure and healing process of their wounds. The WP supplementation also decreased their levels of free radicals and restored their hydroxyproline content; proinflammatory cytokine levels; and expression of BD-1, 2 and 3 in the wounded tissue. CONCLUSION: WP supplementation may be beneficial for improving the healing and closure of diabetic wounds.


Asunto(s)
Antiinfecciosos/farmacología , Diabetes Mellitus Experimental/metabolismo , Expresión Génica/efectos de los fármacos , Proteínas de la Leche/farmacología , Leche/química , Cicatrización de Heridas/efectos de los fármacos , beta-Defensinas/metabolismo , Animales , Antiinfecciosos/aislamiento & purificación , Biomarcadores/metabolismo , Camelus/fisiología , Colágeno/biosíntesis , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/patología , Suplementos Dietéticos , Femenino , Radicales Libres/antagonistas & inhibidores , Radicales Libres/metabolismo , Hidroxiprolina/biosíntesis , Interleucina-6/antagonistas & inhibidores , Interleucina-6/biosíntesis , Masculino , Ratones , Proteínas de la Leche/aislamiento & purificación , Piel/efectos de los fármacos , Piel/lesiones , Piel/metabolismo , Estreptozocina , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesis , Proteína de Suero de Leche , beta-Defensinas/genética
12.
Crit Rev Biochem Mol Biol ; 45(2): 106-24, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20199358

RESUMEN

Posttranslational modifications can cause profound changes in protein function. Typically, these modifications are reversible, and thus provide a biochemical on-off switch. In contrast, proline residues are the substrates for an irreversible reaction that is the most common posttranslational modification in humans. This reaction, which is catalyzed by prolyl 4-hydroxylase (P4H), yields (2S,4R)-4-hydroxyproline (Hyp). The protein substrates for P4Hs are diverse. Likewise, the biological consequences of prolyl hydroxylation vary widely, and include altering protein conformation and protein-protein interactions, and enabling further modification. The best known role for Hyp is in stabilizing the collagen triple helix. Hyp is also found in proteins with collagen-like domains, as well as elastin, conotoxins, and argonaute 2. A prolyl hydroxylase domain protein acts on the hypoxia inducible factor alpha, which plays a key role in sensing molecular oxygen, and could act on inhibitory kappaB kinase and RNA polymerase II. P4Hs are not unique to animals, being found in plants and microbes as well. Here, we review the enzymic catalysts of prolyl hydroxylation, along with the chemical and biochemical consequences of this subtle but abundant posttranslational modification.


Asunto(s)
Procolágeno-Prolina Dioxigenasa/química , Animales , Colágeno/metabolismo , Humanos , Hidroxilación , Hidroxiprolina/biosíntesis , Procolágeno-Prolina Dioxigenasa/fisiología , Prolina/metabolismo , Procesamiento Proteico-Postraduccional , Especificidad de la Especie , Especificidad por Sustrato
13.
J Asthma ; 47(4): 374-80, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20528589

RESUMEN

BACKGROUND: Airway remodeling is one of the cardinal features of asthma and is thought to play a pivotal role in refractory or persistent asthma. Immunoglobulin E (IgE) has a major effect on the pathogenesis of asthma. The aim of this study was to investigate the effects of anti-IgE antibody not only on airway inflammation and bronchial hyperresponsiveness, but also on airway remodeling in a murine model of chronic asthma. METHODS: The authors developed a mouse model of chronic asthma in which ovalbumin (OVA)-sensitized female BALB/c-mice were exposed to intranasal OVA administration twice a week for 3 months. Anti-IgE antibodies were administered intravenously starting on the 38th day and once a month thereafter for 3 months during the intranasal OVA challenge. RESULTS: Mice that were chronically exposed to OVA developed sustained eosinophilic airway inflammation and airway hyperresponsiveness (AHR) to methacholine and showed increased levels of collagen, hydroxyproline, and alpha-smooth muscle actin, as compared with control mice. Treatment with anti-IgE antibody inhibited the development of AHR, eosinophilic inflammation, and airway remodeling. Moreover, anti-IgE antibody treatment reduced the levels of interleukin (IL)-5 and IL-13 in the bronchoalveolar lavage fluids, although it did not affect the levels of IL-10, transforming growth factor-beta, and activin A. CONCLUSION: These results suggest that anti-IgE antibody treatment modulates the airway inflammation and remodeling associated with chronic allergen challenge. The inhibition of inflammation may be related to the regulation of Th2 cytokines. However, the mechanisms underlying the blocking of airway remodeling by anti-IgE antibody remain to be elucidated.


Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Anticuerpos Antiidiotipos/farmacología , Asma/fisiopatología , Inmunoglobulina E/inmunología , Actinas/biosíntesis , Animales , Asma/inmunología , Pruebas de Provocación Bronquial , Líquido del Lavado Bronquioalveolar/inmunología , Enfermedad Crónica , Colágeno/biosíntesis , Citocinas/biosíntesis , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Femenino , Hidroxiprolina/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ovalbúmina
14.
Biochem Biophys Res Commun ; 379(4): 882-6, 2009 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-19133227

RESUMEN

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing L-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-L-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-L-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel L-proline cis-4-hydroxylases converting free L-proline to cis-4-hydroxy-L-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected L-proline cis-4-hydroxylase activity in both proteins. Likewise L-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).


Asunto(s)
Alphaproteobacteria/enzimología , Hidroxiprolina/biosíntesis , Complejo Cetoglutarato Deshidrogenasa/química , Prolina/metabolismo , Alphaproteobacteria/genética , Clonación Molecular , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Cetoglutarato Deshidrogenasa/aislamiento & purificación , Sinorhizobium meliloti/enzimología , Sinorhizobium meliloti/genética , Temperatura
15.
Science ; 182(4114): 825-7, 1973 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-4355681

RESUMEN

An improved procedure was employed for linking ferritin to antibodies against prolyl hydroxylase, the enzyme that synthesizes the hydroxyproline in collagen. By electron microscopy, the enzyme was then found to be localized in cisternae of the rough endoplasmic reticulum of embryonic tendon cells; this indicates that hydroxylation of proline occurs while newly synthesized polypeptides are fed into the cisternae.


Asunto(s)
Ferritinas , Inmunoensayo , Procolágeno-Prolina Dioxigenasa/análisis , Tendones/enzimología , Animales , Embrión de Pollo , Retículo Endoplásmico/enzimología , Pruebas de Inhibición de Hemaglutinación , Hidroxiprolina/biosíntesis , Métodos , Microscopía Electrónica , Procolágeno-Prolina Dioxigenasa/metabolismo , Conejos/inmunología , Tendones/citología
16.
Science ; 152(3718): 92-4, 1966 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-5910019

RESUMEN

Autoradiographs of embryonic cartilage indicated that labeled protein accumnulated intracellularly when the tissue was incubated with tritiated proline, and when the hydroxylation of proline was inhibited by anaerobic conditions or by a chelator for ferrous iron. The labeled protein apparently corresponds to protocollagen. the polypeptide precursor of collagen which serves as a substrate for the enzymatic synthesis of hydroxyproline.


Asunto(s)
Cartílago/metabolismo , Colágeno/biosíntesis , Hidroxiprolina/biosíntesis , Péptidos/metabolismo , Prolina/metabolismo , Animales , Autorradiografía , Isótopos de Carbono , Cartílago/embriología , Quelantes/farmacología , Embrión de Pollo , Técnicas In Vitro , Isótopos de Azufre
17.
Science ; 158(3800): 492-4, 1967 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-6048104

RESUMEN

The hydroxyproline and hydroxylysine in collagen are synthesized by an apparently unique pathway in which proline and lysine are hydroxylated after they are incorporated into a large polypeptide precursor of collagen called protocollagen. When the hydroxylation of protocollagen in isolated tissues is intermittently interrupted, hydroxylation can occur after complete polypeptides are released from ribosomal complexes. Cartilage from chick embryos was incubated with the iron chelator alpha,alpha'-dipyridyl for 2 hours to inhibit protocollagen hydroxylase, and then the inhibition was reversed by transferring the tissues to medium containing ferrous iron and no alpha,alpha'-dipyridyl. "Pulse labeling" of the tissues during these two periods indicated that both the accumulated protocollagen and the polypeptides synthesized after reversal of the inhibition were hydroxylated at the same rate. Even when no measures are taken to inhibit the hydroxylation of protocollagen, most of the hydroxyproline in collagen is probably synthesized after complete protocollagen polypeptides are released from ribosomes.


Asunto(s)
Huesos/metabolismo , Colágeno/biosíntesis , Hidroxiprolina/biosíntesis , Oxigenasas de Función Mixta/metabolismo , Biosíntesis de Péptidos , Péptidos/metabolismo , Ribosomas/metabolismo , Animales , Huesos/citología , Huesos/enzimología , Isótopos de Carbono , Quelantes/farmacología , Embrión de Pollo , Técnicas de Cultivo , Lisina/metabolismo , Prolina/metabolismo , Piridinas/farmacología , Ribosomas/enzimología , Tibia
18.
Bioengineered ; 10(1): 52-58, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-30955438

RESUMEN

Trans-4-hydroxy-L-proline (Hyp) production by Escherichia coli (E. coli) in fermentation is a high-oxygen-demand process. E. coli secretes large amounts of soluble protein, especially in the anaphase of fermentation, which is an important factor leading to inadequate oxygen supply. And acetic acid that is the major by-product of Hyp production accumulates under low dissolved oxygen (DO). To increase DO and achieve high-level Hyp production, soluble protein was hydrolysed by adding protease in Hyp fermentation. The optimal protease, concentration, and addition time were trypsin, 0.2 g/L, and 18 h, respectively. With the addition of trypsin, the soluble protein in Hyp fermentation decreased by 43.5%. The DO could be maintained at 20-30% throughout fermentation. Hyp production and glucose conversion rate were 45.3 g/L and 18.1%, which were increases of 24.1% and 8.4%, respectively. The accumulation of acetic acid was decreased by 52.1%. The metabolic flux of Hyp was increased by 44.2% and the flux of acetate was decreased by 51.0%.


Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Hidroxiprolina/biosíntesis , Oxígeno/química , Prolina/metabolismo , Prolil Hidroxilasas/metabolismo , Ácido Acético/antagonistas & inhibidores , Ácido Acético/metabolismo , Fermentación , Glucosa/agonistas , Glucosa/metabolismo , Humanos , Hidrólisis , Microbiología Industrial/métodos , Cinética , Proteolisis , Solubilidad , Tripsina/química
19.
Eur J Pharmacol ; 852: 77-89, 2019 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-30831079

RESUMEN

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and high mortality, posing a major threat to human health. Increased levels of inflammatory cytokines, reactive oxygen species and coagulation cascade have been extensively reported in IPF. We previously fused Hirudin and human manganese superoxide dismutase (hSOD2) to generate a dual-feature fusion protein, denoted as rhSOD2-Hirudin fusion protein. In this study, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and Hydroxyproline (HYP) assays were used to investigate the effects of rhSOD2-Hirudin protein on thrombin-induced fibroblast proliferation and collagen accumulation in vitro. Subsequently, the mice model of pulmonary fibrosis induced by bleomycin was used for evaluating the anti-inflammatory and anti-fibrotic effects of rhSOD2-Hirudin protein in vivo. Results showed that rhSOD2-Hirudin protein could inhibit the proliferation of fibroblasts and reduce the HYP production in vitro by inhibiting the activity of thrombin. In vivo experiments showed that lung inflammation and fibrosis were significantly decreased in rhSOD2-Hirudin protein-treated mice. Furthermore, rhSOD2-Hirudin protein treatment reduced profibrotic protein and gene expression while reducing the number of inflammatory cells in the lung. In conclusion, rhSOD2-Hirudin protein can effectively attenuate pulmonary fibrosis in vitro and in vivo, mainly by inhibiting the activity of thrombin meanwhile increasing SOD2 levels prevent cells from being damaged by reactive oxygen species, thereby mitigating IPF progression. This study provided important information on the feasibility and efficacy of rhSOD2-Hirudin protein as a novel therapeutic agent for IPF.


Asunto(s)
Bleomicina/farmacología , Hirudinas/genética , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Superóxido Dismutasa/farmacología , Actinas/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hidroxiprolina/biosíntesis , Ratones , Células 3T3 NIH , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Proteínas Recombinantes de Fusión/uso terapéutico , Factor de Crecimiento Transformador beta1/metabolismo
20.
J Biosci Bioeng ; 126(4): 470-477, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29805115

RESUMEN

trans-4-Hydroxy-l-proline (trans-4Hyp) is widely used as a valuable building block for the organic synthesis of many pharmaceuticals such as carbapenem antibiotics. The major limitation for industrial bioproduction of trans-4Hyp is the low titer and productivity by using the existing trans-proline 4-hydroxylases (trans-P4Hs). Herein, three new trans-P4Hs from Alteromonas mediterranea (AlP4H), Micromonospora sp. CNB394 (MiP4H) and Sorangium cellulosum (ScP4H) were discovered through genome mining and enzymatic determination. These trans-P4Hs were introduced into an l-proline-producing chassis cell, and the recombinant strain overexpressing AlP4H produced the highest concentration of trans-4Hyp (3.57 g/L) from glucose in a shake flask. In a fed-batch fermentation with a 5 L bioreactor, the best strain SEcH (pTc-B74A-alp4h) accumulated 45.83 g/L of trans-4Hyp within 36 h, with the highest productivity (1.27 g/L/h) in trans-4Hyp fermentation from glucose, to the best of our knowledge. This study provides a promising hydroxylase candidate for efficient industrial production of trans-4Hyp.


Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosa/metabolismo , Hidroxiprolina/biosíntesis , Oxigenasas de Función Mixta/genética , Alteromonas/enzimología , Proteínas Bacterianas/metabolismo , Reactores Biológicos , Fermentación , Ingeniería Metabólica , Micromonospora/enzimología , Oxigenasas de Función Mixta/metabolismo , Prolina/metabolismo
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